Voges Proskauer (VP) Test – Principle, Procedure, Results

What is Voges Proskauer (VP) Test?

Voges–Proskauer (VP) test is a biochemical test used to detect acetylmethylcarbinol (acetoin) produced during glucose fermentation. It is the process by which organisms ferment glucose through the butylene glycol pathway. This test is included in IMViC series of biochemical tests and it is used for identification of Gram-negative bacilli .

In this test, the organism is inoculated into glucose phosphate broth and incubated. During fermentation, if the organism follows butylene glycol pathway, acetoin is formed as an intermediate product. After incubation, two reagents are added i.e. alpha-naphthol (Barritt’s reagent A) and potassium hydroxide (KOH) (Barritt’s reagent B). In the presence of oxygen and strong alkali, acetoin is oxidized to diacetyl. The diacetyl reacts with guanidine compounds present in the peptone of the medium and a red coloured complex is formed. This is referred to as positive VP reaction.

If acetoin is not produced, no red colour is developed. The medium may remain yellow or sometimes it shows copper or brownish-green colour. This indicates negative result. It is mainly used to differentiate Escherichia coli (VP negative) from Klebsiella and Enterobacter species (VP positive). Thus, VP test is important for differentiation of enteric organisms.

Definition of Voges Proskauer (VP) Test

The Voges-Proskauer (VP) Test is a biochemical assay used to detect the ability of certain bacteria to metabolize glucose into a neutral intermediate product called acetoin during fermentation. Positive results are indicated by the development of a red or pink color after the addition of specific reagents. This test aids in the differentiation and identification of Gram-negative bacteria, particularly within the Enterobacterales order.

Objectives of Voges–Proskauer (VP) Test

• To detect the production of acetylmethylcarbinol (acetoin) from glucose fermentation by the organism.
• To determine whether the bacteria utilize butylene glycol (butanediol) pathway for fermentation of glucose.
• To differentiate Gram-negative bacilli, especially members of Enterobacteriaceae family.
• To separate VP negative organisms such as Escherichia coli from VP positive organisms such as Klebsiella and Enterobacter species.
• To characterize and differentiate closely related enteric bacteria based on their neutral end products formation.

Principle of Voges Proskauer (VP) Test

The principle of Voges–Proskauer (VP) test is based on the detection of acetylmethylcarbinol (acetoin) produced during glucose fermentation by butylene glycol pathway. It is the process in which bacteria ferment glucose and form neutral end products such as acetoin and 2,3-butanediol. This acetoin is the key intermediate detected in this test.

After incubation of the organism in glucose broth, alpha-naphthol and strong alkali (40% potassium hydroxide) are added. Under alkaline condition and in the presence of atmospheric oxygen, the acetoin produced is oxidized to diacetyl. The reaction is as follows– acetoin is converted into diacetyl in presence of KOH and oxygen.

The alpha-naphthol acts as catalyst and colour intensifier in this reaction. The diacetyl formed then reacts with guanidine containing compounds present in the peptone of the medium and a pinkish-red or cherry-red coloured complex is formed. This is referred to as positive VP reaction.

If acetoin is absent, diacetyl is not formed and no red colour is produced. The medium may remain yellow or sometimes develops copper or brownish-green colour. This indicates negative VP test. Thus, the formation of red colour confirms the presence of acetoin in the medium.

Requirements for VP Test

• Culture medium– MR-VP broth (Glucose Phosphate Broth).
• Reagents– Barritt’s reagent A (5% alpha-naphthol solution) and Barritt’s reagent B (40% potassium hydroxide or sodium hydroxide solution).
• Test tubes, droppers, inoculating loop, incubator, Bunsen burner, weighing balance and autoclave.
• Test organism from pure culture.
• Control organisms– Klebsiella pneumoniae (positive control) and Escherichia coli (negative control).

Control Organisms for Voges–Proskauer (VP) Test

– Positive control– Enterobacter aerogenes (ATCC 13048).

– Positive control– Enterobacter cloacae (ATCC 23355).

– Positive control– Klebsiella pneumoniae (ATCC 13883).

– Negative control– Escherichia coli (ATCC 25922).

Procedure of of Voges Proskauer (VP) Test

1. A pure culture of the test organism (18–24 hours old) is taken with the help of sterile inoculating loop and inoculated into MR-VP broth (Glucose Phosphate Broth).
2. The inoculated broth is incubated aerobically at 35–37°C for 24–48 hours.
3. After incubation, about 1–2 ml of the broth is transferred into a clean sterile test tube.
4. Barritt’s reagent A (5% alpha-naphthol) is added to the broth. The reagents are added in proper order otherwise false negative reaction may occur.
5. Then Barritt’s reagent B (40% potassium hydroxide) is added to the tube.
6. The tube is shaken vigorously for about 30 seconds to 1 minute to allow entry of atmospheric oxygen. This step is important for oxidation of acetoin.
7. The tube is allowed to stand undisturbed for 15–30 minutes for colour development.
8. The upper layer of the medium is observed. Development of pink-red or cherry-red colour indicates positive VP test, whereas yellow, copper or brownish-green colour indicates negative result. If no colour is observed at 30 minutes, it is allowed to stand up to one hour before final interpretation.

Results and Interpretation of Voges Proskauer (VP) Test

Positive result– Development of pinkish-red, cherry-red or red-brown colour at the surface of the medium indicates positive VP test. It confirms that the organism ferment glucose through butylene glycol pathway and acetoin is produced.

Negative result– Absence of red colour indicates negative reaction. The medium remains yellow or amber in colour or it may turn brownish-green. It shows that acetoin is not produced by the organism.

Copper colour (Negative)– Development of copper or bronze colour is interpreted as negative result. This colour is produced due to reaction between potassium hydroxide and alpha-naphthol in absence of diacetyl.

Weak positive result– Appearance of rust or orange-red colour indicates weakly positive reaction. It occurs when the organism produce low amount of acetoin.

Voges Proskauer (VP) Test Results of Some Common Bacteria

VP-Positive Organisms These bacteria ferment glucose via the butanediol pathway to produce acetoin:

• Enterobacter species (e.g., E. aerogenes, E. cloacae, E. intermedium)
• Klebsiella species (e.g., K. pneumoniae, K. oxytoca, K. planticola, K. terrigena)
• Serratia species (e.g., S. marcescens, S. liquefaciens)
• Staphylococcus species (e.g., S. aureus, S. epidermidis, S. saprophyticus, S. intermedius)
• Micrococcus species (e.g., M. luteus, M. kristinae)
• Listeria species (e.g., L. monocytogenes)
• Certain Vibrio species (e.g., V. cholerae biotype El Tor, V. alginolyticus, V. anguillarum, V. costicola, V. damsela)
• Certain Streptococcus species (e.g., S. mutans, S. agalactiae, S. pyogenes, S. bovis, S. salivarius)
• Aeromonas species (e.g., A. hydrophilia, A. salmonicida subsp. masoucida, A. veronii, A. sobria)
• Enterococcus fecalis
• Proteus myxofaciens

VP-Negative Organisms These bacteria typically ferment glucose through the mixed-acid pathway or do not produce acetoin:

• Escherichia species (e.g., E. coli)
• Salmonella species (e.g., S. enterica)
• Shigella species (e.g., S. sonnei)
• Citrobacter species (e.g., C. freundii)
• Yersinia species
• Edwardsiella species
• Providencia species
• Morganella species (e.g., M. morganii)
• Proteus vulgaris
• Certain Vibrio species (e.g., V. parahaemolyticus, V. vulnificus, V. furnissii, V. fluvialis)
• Certain Streptococcus species (e.g., S. mitis, S. vestibularis, S. sanguis)
• Certain Klebsiella subspecies (K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. rhinoscleromatis)

VP-Variable Organisms These bacteria can yield either positive or negative results depending on specific factors like temperature or incubation time:

• Hafnia alvei: Highly temperature-dependent. It typically tests positive when grown at 25°C to 30°C, but often tests negative at standard clinical incubation temperatures of 35°C to 37°C.
• Proteus mirabilis: Often gives a delayed positive reaction, with some strains testing positive and others negative.

Precautions of Voges Proskauer (VP) Test

• A light inoculum should be used for the test. Heavy inoculation may inhibit the growth and may give invalid result.
• The reagents must be added in proper order. Alpha-naphthol (Reagent A) is added first and then potassium hydroxide (Reagent B) is added. Reversing the order may produce false positive or false negative reaction.
• Proper aeration is required after adding the reagents. The tube is shaken vigorously to introduce atmospheric oxygen which is necessary for oxidation of acetoin to diacetyl.
• The result should be read within one hour after adding the reagents. Prolonged standing may lead to false interpretation due to reaction between the reagents.
• Over incubation of the broth should be avoided. Incubation for more than 72 hours may result in false negative reaction as acetoin may be further metabolized by the organism.
• The reagents should be added in correct amount. Excess potassium hydroxide may mask the reaction or produce weak colour.
• Fresh reagents should be used and they should be stored properly in dark bottles at 4–8°C. Potassium hydroxide is highly caustic and care should be taken to avoid contact with skin and eyes.
• Copper, bronze or brownish-green colour should not be considered as positive result. This colour indicates negative VP reaction.

Uses of Voges Proskauer (VP) Test

• It is used for differentiation of Gram-negative bacilli, especially members of Enterobacteriaceae family based on their ability to produce neutral end products from glucose fermentation.
• It helps in separation of closely related enteric organisms such as Escherichia coli (VP negative) from Klebsiella and Enterobacter species (VP positive).
• It is used in identification of Vibrio species. VP positive Vibrio cholerae and Vibrio alginolyticus can be differentiated from VP negative Vibrio parahaemolyticus and Vibrio vulnificus.
• It is also used for characterization of other bacterial groups such as Actinobacteria and some Gram-positive organisms which produce acetoin like Staphylococci, Micrococci, Listeria and Viridans Streptococci.
• It is applied in food and dairy industry for detection of acetoin and diacetyl which are important flavour components in butter, cream, cheese, wine and beer.

Limitations of Voges Proskauer (VP) Test

• It is not a confirmatory test. The VP test is only an aid in identification and it cannot be used alone for complete identification of bacterial species. It should be interpreted along with other biochemical and morphological tests.
• The reagents must be added in exact order and proper amount. Alpha-naphthol is added first followed by potassium hydroxide. Reversing the order or adding excess KOH may give false negative or weak positive reaction.
• The result is time dependent. The test should be read within one hour after addition of reagents. Reading after long time may produce false positive result due to reaction between the reagents forming copper like colour.
• Prolonged incubation of the broth culture may affect the result. Incubation beyond 48–72 hours may lead to false negative or weak reaction as acetoin produced may be further broken down by the organism.
• Heavy inoculum may interfere with the reaction. Very turbid culture can inhibit proper growth and may give invalid result.
• Proper aeration is necessary for the reaction. If the tube is not shaken properly after adding the reagents, oxidation of acetoin to diacetyl may not occur and false negative result is obtained.
• Some organisms show variable result depending on temperature and strain variation. Certain bacteria may show positive reaction at lower temperature and negative at higher temperature. Variation in media composition may also affect the result.

FAQ

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